Bacteria are prevalent throughout the human body, residing at all interfaces with the external environment. These microbes, which outnumber human cells ten to one, are highly physiologically active and known to influence the health of their host. Although most studies to date have focused on microbes of the skin, oropharynx, gastrointestinal tract, and vagina, there is now an understanding that the lungs are not sterile, as was once believed. In fact, evidence shows that microbial burden and diversity in the lungs differs between healthy individuals and those with lung disease. Additionally, microbial composition in the lungs has been linked to respiratory disease severity. Since it is known that microbial communities in the gut can metabolize environmental toxicants, stimulate host inflammatory response, and increase risk of host infection, it is likely that respiratory microbes also play an important role in human health. A major challenge to researching the respiratory microbiome is the lack of a well-established, non-invasive measure in the lungs. Bronchoalveolar lavage is the most direct approach to measure the lungs, but it is an invasive procedure performed under sedation at the hospital. This makes it too burdensome on participants and cost-prohibitive for large clinical and epidemiology studies. To overcome these limitations, the aim of this exploratory study is to systematically investigate exhaled breath condensate as a novel, non-invasive medium to characterize the respiratory microbiome. Because exhaled breath condensate may reflect several inter-connected biological compartments, we plan to characterize the microbial communities of concurrent samples of exhaled breath condensate, bronchoalveolar lavage fluid, oral wash, nasal swab, fecal, and inspired air samples collected from 18 healthy individuals. Specifically we plan to: 1) assess how well the microbial communities in exhaled breath condensate reflect the microbial communities in the lungs and 2) identify the origins (mouth, nose, gut, inspired air) of any microbial communities in exhaled breath condensate not present in the lungs. The ability to characterize the lung microbiome using exhaled breath condensate, alone or paired with other non-invasive samples will open the door to large-scale population-based epidemiology studies that are currently cost-prohibitive and logistically infeasible. We plan to follow this work with future studies of the respiratory microbiome's role in processing inhaled environmental pollutants, predicting survival among patients with severe lung disease, and as a diagnostic for clinical use. Thus, this work is expected to lead to substantial improvements in our understanding of the role of the respiratory microbiome on human health.